Information
This kit is recommended for users who:
- want to achieve raw read sequencing modal accuracy of Q20+ (99%+)
- want to optimise their sequencing experiment for accuracy
- wish to multiplex samples to reduce price per sample
- need a PCR-free method of multiplexing to preserve additional information such as base modifications
- require control over read length
- are interested in utilising upstream processes such as size selection or whole genome amplification
This is an Early Access product
For more information about our Early Access programmes, please see this article on product release phases..
The Native Barcoding Kit 96 features:
| Feature | Property |
|---|
| Preparation time | 140 minutes |
| Input requirement | 400 ng per sample of gDNA
200 fmol (130 ng for 1 kb amplicons) per sample of amplicons |
| PCR Required | No |
| Fragmentation | Optional |
| Read length | Equal to fragment length |
| Read type produced | 1D |
| Associated protocols | • Ligation sequencing gDNA - native barcoding (SQK-NBD112.96)
• Native Barcoding Kit 96 with amplicons (SQK-NBD112.96) |
| Pack size | 12 reactions
Options include: 3 libraries containing 96 barcodes, 6 libraries containing 48 barcodes or 12 libraries containing 24 barcodes |
| Stability | Shipped at 2–8°C
Long-term storage -20°C
Oxford Nanopore Technologies deem the useful life of the product to be 3 months from receipt by the customer |
Barcoding or multiplexing is useful when the amount of data required per sample is less than the total amount of data that can be generated from a single flow cell: it allows a user to pool multiple samples and sequence them together, making more efficient use of the flow cell.
Multiplexing samples onto one flow cell can reduce the cost per sample for a user. Below are some worked examples:
| No Barcodes | 24 barcodes | 48 barcodes | 96 barcodes |
|---|
| Flow cell price | $500 | $500 | $500 | $500 |
| Library price | $99 | $99 | $99 | $99 |
| Barcode price | - | $75 | $150 | $300 |
| Price per sample | $599 | $28 | $15 | $9.36 |
The Native Barcoding Kit 96 is a standalone kit providing 96 unique barcodes to enable PCR-free multiplexing of dsDNA samples such as gDNA and amplicons.
The library preparation method is similar to the Ligation Sequencing Kit protocol; gDNA ends are repaired (note: DNA repair is skipped when using amplicon input) and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.
The kit is optimised to achieve sequencing accuracies of over 99% (Q20+). For highest data yields, we recommend starting with 400 ng per sample for both R9.4.1 and R10.4 flow cells. Starting with lower amounts of input material, or impure samples, can affect library preparation efficiency and can reduce sequencing output.
This Native Barcoding Kit (Kit 12) includes the Adapter Mix II H (AMII H), with three key features: Firstly, the adapter is loaded with an updated sequencing enzyme enabling accuracies of over 99% (Q20+). Secondly, the adapter is higher capture, enabling lower flow cell loading amounts. Finally, the adapter contains the fuel fix technology enabling users to run long experiments without the need for fuel addition during the run.
If your experiment requires long reads, it is recommended to start with full-length gDNA, and fragmentation/shearing is neither advised nor required. Please note: if long reads are required, high molecular weight DNA should be used as input. To determine purity, we suggest using the Nanodrop to measure the A260/280 and A260/230 ratios and we recommend that the sample should meet the following criteria:
- A260/280 = 1.8
- A260/230 = 2.0-2.2
The Native Barcoding Kit 96 is compatible with upstream processes such as whole genome amplification (for applications where under 1 ng of sample is available) and size selection (for enrichment of specified fragment lengths, using the BluePippin, for example). When size selecting, we recommend increasing the amount of input used, as size selection can be a wasteful process.
Deconvolution of barcoded sequencing data is supported by MinKNOW, Guppy and EPI2ME which classify the barcode sequence and sort reads into corresponding folders.
Further considerations:
Starting with lower amounts of input material, or impure samples can affect library preparation efficiency and lower sequencing throughput. PCR can be used to generate more input material in cases where sample amount is limiting.
Shipping and logistics:
Flow cells and kits are shipped together in an environmentally friendly temperature-controlled shipping box.
Products are shipped to customers within the USA and EU Monday to Thursday. Shipments to Canada, Norway, Korea and Japan are expedited Monday to Wednesday; with Australia and New Zealand leaving our warehouses on a Friday. Shipments to the rest of the world are made on Mondays to allow the full working week for packages to arrive.
The delivery charges are calculated when a quote is raised or during checkout. Once an order is made, the delivery ID and delivery information can be tracked in the Store.
Workflow
The library preparation method is similar to the Ligation Sequencing Kit protocol; DNA ends are repaired (note: DNA repair is skipped when using amplicon input) and dA-tailed using the NEBNext End Repair/dA-tailing module, and then a unique dT-tailed barcode adapter is ligated on the dA-tailed template. Barcoded samples are then pooled together. Each barcode adapter also has a cohesive end and this is used as a hook to ligate to the supplied sequencing adapters.
What's in the box
The Native Barcoding Kit 96 contains 96 unique barcodes and sufficient reagents for 12 reactions (options include: 3 libraries containing 96 barcodes, 6 libraries containing 48 barcodes, or 12 libraries containing 24 barcdoes)
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|
| Adapter Mix II H | AMII H | Green | 2 | 40 |
| Sequencing Buffer II | SBII | Red | 2 | 500 |
| Loading Beads II | LBII | Pink | 2 | 360 |
| Loading Solution | LS | White cap, pink label | 2 | 400 |
| Elution Buffer | EB | Black | 1 | 500 |
| AMPure XP Beads | AXP | White | 1 | 6,000 |
| L Fragment Buffer | LFB | White | 1 | 7,500 |
| S Fragment Buffer | SFB | White | 1 | 7,500 |
| EDTA | EDTA | Clear | 1 | 700 |
| Flush Buffer | FB | White | 1 | 15,500 |
| Flush Tether | FLT | Purple | 1 | 400 |
Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Compatibility
The Native Barcoding Expansion Kit 96 can be used together with:
Kits
- Sequencing Auxiliary Vials (EXP-AUX002)
- Flow Cell Wash Kit (EXP-WSH004)
- Native Barcoding Expansion (EXP-NBD112)
Flow cells
- FLO-MIN106D
- FLO-MIN112
- FLO-PRO002
- FLO-PRO112
Devices
- MinION Mk1B
- MinION Mk1C
- GridION
- PromethION
